1. Field of the Invention
The present invention relates to a method for optically resolving a racemic xcex1-substituted heterocyclic carboxylic acid (hereinafter referred to as xe2x80x9cxcex1-HCCAxe2x80x9d). More particularly, the present invention pertains to a method for optically resolving a racemic xcex1-HCCA using an enzyme catalyst with enantioselectivity.
2. Description of the Prior Art
Divided into optical isomers, R- and S-forms, tetrahydro-2-furoic acid (hereinafter referred to as xe2x80x9cTHFAxe2x80x9d), a kind of xcex1-HCCA, is an important chiral building block which has various applications in chemistry. Of the optical isomers, R-(+)-THFA is used as a side chain intermediate for the synthesis of penem type antibiotics while S-(xe2x88x92)-THFA is useful as a chiral intermediate for organic synthesis. Therefore, THFA is different in use in R form and S form thereof. However, because THFA is obtained in the form of a racemic mixture when chemically synthesized, additional processes are required to separate THFA into enantiomers thereof: R and S forms.
Optical resolution has been usually used to divide racemic THFA into R- and S-forms thereof. In 1983, Belanger successfully separated THFA racemate into enantiomers thereof by use of brucine and ephedrine as resolving agents (Can. J. Chem., 61, 1383 (1983)). However, the resolving agents are not economical because of their being very expensive. Another problem with this process is that its products are low in enantiomeric excess value.
Japanese Pat. Laid-Open Publication No. 89-216983 discloses the use of a chiral amine (1-(4-halogenophenyl)ethylamine) as a resolving agent, in which diastereomer salts are prepared from R,S-THFA and optically resolved. This method is also economically unfavorable owing to the high price of the chiral amine. Additionally, only low production yields can be obtained because the amount of R,S-THFA to be added in the early reaction is limited to as low as 4 mmol. Furthermore, the chiral THFA finally obtained is poor in enantiomeric excess value.
A method different from optical resolving methods is found in Japanese Pat. Laid-Open Publication. No. 97-71576 which refers to synthesizing R- or S-THFA by treating R- or S-THFA salts with hydrogen halide.
It has been well known for some time that optical resolution of racemates could be achieved by use of enzymes, such as esterases, lipases, and proteases, as enzyme catalysts to enantioselectively hydrolyze one of the two enantiomers present. For example, U.S. Pat. No. 5,928,933 discloses an enzyme with an enantiomeric excess of 95% as a result of extensive experiments for reaction specificity of 44 enzymes, including proteases, lipases and esterases. The enzyme catalyst is very useful for the separation of enantiomeric racemates, but because the selectivity for enantiomers and the optical purity of products may vary depending on the choice of enzymes and chemical structures of their substrates, intensive efforts are required to find combinations of enzymes suitable for substrates. Especially, nowhere is found a method for optical resolution of xcex1-HCCA using an enzyme.
Therefore, there remains a need for an enzymatic optical resolution method that can divide racemic xcex1-HCCA into R- and S-form economically and easily.
Leading to the present invention, the intensive and through research on the optical resolution of xcex1-HCCA, conducted by the present inventors aiming to develop optically highly pure xcex1-HCCA by an economical procedure, resulted in the finding that some of microorganism- or animal-derived hydrolyzing enzymes may enantioselectively hydrolyze the ester functionality of particular optical isomers of xcex1-HCCA at high efficiency.
Therefore, it is an object of the present invention to overcome the above problems encountered in prior arts and to provide a method for optically resolving a racemic xcex1-HCCA using an enzyme, which is economically favorable.
In one aspect of the present invention, there is provided a method for optically resolving a racemic xcex1-HCCA, comprising the steps of:
reacting a racemic xcex1-HCCA with alcohol to give a racemic xcex1-HCCA ester represented by the following chemical formula 1: 
wherein R1 is selected from the group consisting of substituted or unsubstituted alkyl or alkenyl containing 1 to 6 carbon atoms, benzyl, cycloalkyl containing 3 to 6 carbon atoms, substituted or unsubstituted arylalkyl, and substituted or unsubstituted heteroarylalkyl, X represents O, S or Nxe2x80x94H, and n is an integer of 1 to 3;
optically resolving the racemate of the formula 1 by use of an enzyme with enantioselectivity to hydrolyze either R-form or S-form of the ester racemate thereby producing a R-form or S-form of xcex1-HCCA and a counter enantiomeric form of xcex1-HCCA ester thereto, said enzyme existing as a powder or an aqueous solution;
extracting the unhydrolyzed xcex1-HCCA ester with an organic solvent; and
subjecting the extracted xcex1-HCCA ester in an organic solvent to hydrogenation under a constant hydrogen partial pressure at a constant temperature in the presence of a palladium catalyst on carbon (Pd/C).
In another aspect of the present invention, there is provided a method for optically resolving a racemic xcex1-HCCA, comprising the steps of:
reacting a racemic xcex1-HCCA with alcohol to give a racemic xcex1-HCCA ester represented by the chemical formula 1;
optically resolving the racemate of the formula 1 by use of an enzyme with enantioselectivity to hydrolyze either R-form or S-form of the ester racemate, thereby producing a R-form or S-form of xcex1-HCCA and a counter enantiomeric form of xcex1-HCCA ester thereto, said enzyme existing as a powder or an aqueous solution;
extracting the unhydrolyzed xcex1-HCCA ester with an organic solvent; and
treating the extracted xcex1-HCCA ester with a non-enantioselective enzyme in an aqueous solution at a constant pH and temperature, said enzyme existing as a powder or an aqueous solution.
The present invention is characterized by the enantioselective hydrolysis of esters of racemic xcex1-HCCA by an enzyme to produce a certain enantiomeric form of xcex1-HCCA and a counter enantiomeric form of the esters of xcex1-HCCA, at once. The separation of the hydrolyzed xcex1-HCCA and the remaining esters of xcex1-HCCA can be achieved by use of an organic solvent. The unhydrolyzed enantiomeric form of xcex1-HCCA ester can be hydrolyzed in the presence of non-enantioselective enzyme or be hydrogenated in the presence of a palladium catalyst on carbon (Pd/C) to obtain the corresponding xcex1-HCCA thereto.
In detail, a racemic mixture of xcex1-HCCA is reacted with an alcohol at an equivalent amount to produce a racemic mixture of an xcex1-HCCA ester, which is then enantioselectively hydrolyzed at a constant temperature and pH in an aqueous solution in the presence of an enzyme with enantioseletivity. As a result, the reaction produces an R- or S-form xcex1-HCCA, along with the ester of xcex1-HCCA which has an enantiomeric form counter to that of the hydrolyzed xcex1-HCCA. After completion of the enantioselective hydrolysis, addition of an organic solvent extracts the ester of xcex1-HCCA thereinto, leaving the xcex1-HCCA in the aqueous phase only. Removal of the organic solvent from the organic phase results in acquisition of an optically pure S- or R-form of xcex1-HCCA ester. Poor in optical purity, the xcex1-HCCA remaining in the aqueous solution may be increased in purity through a purification process using, for example, a column, or may be reused as a starting material in the present invention.
In accordance with a preferred embodiment of the present invention, THFA, which belongs to an (xcex1-HCCA, is used as a starting material and after optical resolution, R- or S-form of THFA can be obtained at a high enantiomeric excess. Aside from THFA, all materials falling within the scope of xcex1-HCCA, for example, proline and tetrahydrothiopen-2-carboxylic acid can be optically resolved in accordance with the present invention.
Useful in the present invention are linear or branched C1-C6 alcohols, aromatic alcohols, C3-C6 cycloalkyl alcohols, substituted or unsubstituted arylalkyl alcohols, and substituted or unsubstituted heteroarylalkyl alcohols. Preferred are linear alcohols containing 4 or more carbon atoms or aromatic alcohols, when consideration is taken of reaction time and optical purity.
For use in the optical resolution of racemic xcex1-HCCA ester, the enzyme must enantioselectively hydrolyze the ester functionality of a particular isomer of the racemate. Preferably, the enzyme is selected from the group consisting of microorganism- or animal-derived lipases, proteases, and esterases. Depending on enzymes and chemical structures of substrates, the conformation of the xcex1-HCCA hydrolyzed is determined. Also, the selectivity for enantiomers and the enantiomeric excess value of the product are dependent on enzyme and substrate. Such an enantioselective enzyme, when used, may be in a form of a powder or an aqueous solution. The enzyme is preferably used in an amount of 0.1 to 100 parts by weight based on 100 parts by weight of the xcex1-HCCA ester. For example, if the enzyme is used at an amount less than 0.1 part by weight, the hydrolysis may require excessive time to complete. On the other hand, an enzyme amount exceeding 100 parts by weight increases the production cost.
The enzymatic reaction is optimally carried out at 0-60xc2x0 C. and pH 4-12. As for the organic solvent to extract the remaining enantiomeric xcex1-HCCA ester, it is preferably selected from the group consisting of ethyl acetate, dichloromethane, chloroform, carbon tetrachloride, toluene and mixtures thereof.
Turning now to the reduction of the prepared enantiomeric xcex1-HCCA ester to a corresponding conformation of xcex1-HCCA, two methods are provided for converting an optically pure S- or R-form of xcex1-HCCA ester to a corresponding conformation of xcex1-HCCA with high enantiomeric excess ( greater than 99%), in accordance with the present invention.
First, a solution of the recovered S- or R-form of the xcex1-HCCA ester in an organic solvent is subjected to hydrogenation in the presence of palladium catalyst on carbon (Pd/C) under a constant hydrogen partial pressure at a constant temperature.
In this regard, the palladium catalyst is preferably used in an amount of 0.1-30% by weight and more preferably in an amount of 0.5-10% by weight. For example, an amount less than 0.1% by weight is insufficient to perform the hydrogenation. On the other hand, an amount larger than 30% by weight has negative influence on the production cost. The catalytic hydrogenation of the enantiomeric xcex1-HCCA ester is preferably carried out at a hydrogen partial pressure of 1 to 10 bars and more preferably at a hydrogen partial pressure of 1 to 5 bars. For example, the hydrogenation, when being carried out at a hydrogen partial pressure less than 1 bar, is significantly deteriorated in efficiency. On the other hand, a hydrogen partial pressure larger than 10 bars results in formation of a lot of side products. Other conditions are set at 1 to 20 hours and preferably at 1 to 8 hours for reaction time and at 0 to 70xc2x0 C. and preferably at 20 to 40xc2x0 C. for reaction temperature.
Of THFA esters, THFA benzyl ester can allow THFA to be recovered therefrom with very high ease. For example, a solution of THFA benzyl ester in an organic solvent is hydrogenated at a constant temperature under a constant hydrogen partial pressure to produce THFA and toluene and simple vacuum distillation removes the toluene, leaving THFA only.
An alternate method is to use an enzyme capable of non-enantioselectively hydrolyzing the ester functionality of the recovered optical isomer of the xcex1-HCCA ester with maintenance of a constant pH and temperature. The enzyme, when used in the non-enantioselective hydrolysis, may be in a form of a powder or an aqueous solution.
After completion of the enzymatic hydrolysis, the aqueous phase is collected and adjusted to pH 2-3 with HCl. Several extractions of the acidified aqueous phase with an organic solvent yield a highly pure S- or R-form of xcex1-HCCA.